Heat-induced aggregation of XRCC5 (Ku80) in nontolerant and thermotolerant cells

B. D. Beck, Joseph Dynlacht

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

XRCC5 (also known as Ku80) is a component of the DNA-dependent protein kinase (DNA-PK), existing as a heterodimer with G22P1 (also known as Ku70). DNA-PK is involved in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair, and kinase activity is dependent upon interaction of the Ku subunits with the resultant DNA ends. Nuclear XRCC5 is normally extractable with non-ionic detergent; it is found in the soluble cytoplasmic fraction after nuclear isolation with Triton X-100. In this study, we found that heating at 45.5°C causes a decreased extractability of XRCC5 from the nuclei of human U-1 melanoma or HeLa cells. Such decreases in extractability are indicative of protein aggregation within nuclei. Recovery of extractability of XRCC5 to that of unheated control cells was observed after incubation at 37°C after heat shock. The decrease in extractability and the kinetics of recovery were dependent on dose, although the decrease in extractability reached a plateau after heating for 15 min or more. Thermotolerant U-1 cells also showed decreased extractability of XRCC5, but to a lesser degree compared to nontolerant cells. When a comparable initial reduction of extractability of XRCC5 was induced in both thermotolerant and nontolerant cells, the kinetics of recovery was nearly identical. The kinetics of recovery of the extractability of XRCC5 was different from that of total nuclear protein in nontolerant cells; recovery of extractability of XRCC5 occurred faster initially and returned to the level in unheated cells faster than total nuclear protein. Similar results were obtained for thermotolerant cells, with differences between the initial recovery of the extractability of XRCC5 and total protein being particularly evident after longer heating times. Heat has been shown to inactivate XRCC5. We speculate that inactivation of XRCC5 after heat shock results from protein aggregation, and that changes in XRCC5 may, in part, lead to inhibition of DSB repair through inactivation of the NHEJ pathway.

Original languageEnglish
Pages (from-to)767-774
Number of pages8
JournalRadiation Research
Volume156
Issue number6
StatePublished - 2001

Fingerprint

Hot Temperature
heat
proteins
recovery
cells
DNA-Activated Protein Kinase
Heating
deoxyribonucleic acid
Nuclear Proteins
nuclear proteins
DNA
kinetics
protein kinases
strands
deactivation
DNA End-Joining Repair
heating
heat stress
inactivation
shock

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Radiology Nuclear Medicine and imaging
  • Biophysics
  • Radiation

Cite this

Heat-induced aggregation of XRCC5 (Ku80) in nontolerant and thermotolerant cells. / Beck, B. D.; Dynlacht, Joseph.

In: Radiation Research, Vol. 156, No. 6, 2001, p. 767-774.

Research output: Contribution to journalArticle

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